REVIEWS

Synovial tissue research:

a state‑of‑the-art review
Carl Orr1, Elsa Sousa2, David L. Boyle3, Maya H. Buch4, Christopher D. Buckley5,
Juan D. Cañete6, Anca I. Catrina7, Ernest H. S. Choy8, Paul Emery4, Ursula Fearon9,
Andrew Filer5, Danielle Gerlag10,11, Frances Humby12, John D. Isaacs13, Søren A. Just14,
Bernard R. Lauwerys15, Benoit Le Goff16, Antonio Manzo17, Trudy McGarry9,
Iain B. McInnes18, Aurélie Najm16, Constantino Pitzalis12, Arthur Pratt13, Malcolm Smith19,
Paul P. Tak10,20, Rogier Thurlings21, João E. Fonseca2 and Douglas J. Veale1
Abstract | The synovium is the major target tissue of inflammatory arthritides such as rheumatoid
arthritis. The study of synovial tissue has advanced considerably throughout the past few decades
from arthroplasty and blind needle biopsy to the use of arthroscopic and ultrasonographic
technologies that enable easier visualization and improve the reliability of synovial biopsies.
Rapid progress has been made in using synovial tissue to study disease pathogenesis, to stratify
patients, to discover biomarkers and novel targets, and to validate therapies, and this progress
has been facilitated by increasingly diverse and sophisticated analytical and technological
approaches. In this Review, we describe these approaches, and summarize how their use in
synovial tissue research has improved our understanding of rheumatoid arthritis and identified
candidate biomarkers that could be used in disease diagnosis and stratification, as well as in
predicting disease course and treatment response.

Chronic inflammatory arthritides comprise a heterogene-
Fibroblast-like synoviocytes
(FLSs). Also known as type B ous group of diseases that are characterized by inflamma-
synovial lining cells, FLSs tion of the synovium, which is often accompanied by the
account for the majority of destruction of adjacent cartilage and bone. Inflammation
cells in the synovial lining layer. is characterized by synovial neovascularization, stromal
proliferation and leukocyte extravasation1. For the pur-
pose of this Review, we focus on rheumatoid arthritis
(RA), owing to its prevalence and the fact that this dis-
ease is the most extensively studied and most common
cause of synovitis. RA is usually persistent and progres-
sive, and leads to joint damage, disability and deformity if
left untreated. The disease is associated with a reduction
in quality of life, as well as with decreased longevity, and
represents an important burden on health care spending2–4.
Within the past two decades, several considerable
1
Centre for Arthritis and advances have been made in the treatment of inflamma-
Rheumatic Disease, tory arthritides in general, and particularly in the treat-
University College Dublin, ment of RA. However, further progress is needed. The
Dublin Academic Medical
patterns of clinical response to treatment are remarkably
Centre, St. Vincent’s
University Hospital, Elm Park, similar for agents with different targets, and this finding
Dublin 4, Ireland. challenges our current understanding of disease mecha-
Correspondence to D.J.V.  nisms. In addition, despite the aforementioned unprece-
douglas.veale@ucd.ie dented progress, a substantial proportion of patients with
doi:10.1038/nrrheum.2017.115 RA still do not achieve a state of low disease activity or
Published online 13 Jul 2017 remission following treatment5,6.
The main challenges in biomedicine and transla-
tional research in RA are early diagnosis, personalized
medicine and the development of meaningful outcome
assessments7. A logical hypothesis is that each of these
aims can be facilitated by the identification and devel-
opment of appropriate biomarkers. However, although
peripheral blood biomarkers such as rheumatoid factor
and anti-citrullinated protein antibodies (ACPAs) have
been shown to be relatively specific and might predict the
development of RA in asymptomatic individuals8, they
are reportedly found in only 70–80% of patients with
RA9. Indeed, beyond rheumatoid factor and ACPAs, our
repertoire of blood biomarkers to assist with diagnosis
and to provide insights into disease progression and
response to therapy is currently extremely limited10,11.
As the synovium is the principal target of inflamma-
tion in RA, and the resident fibroblast-like synoviocytes
(FLSs) are implicated in the pathogenesis of synovitis, one
promising approach could be to search for biomarkers in
inflamed synovial tissue. Using a combination of estab-
lished methodologies, together with new high-throughput
omics technologies that have the capability to examine the
expression of genes and their products on a scale never
before possible (BOX 1), a new opportunity awaits in the
search for these biomarkers. This Review summarizes

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Key points how synovial tissue research has advanced our under-
standing of RA, contributed to progress in addressing
• Synovial tissue is the target tissue for autoimmune arthritides such as rheumatoid key challenges in the field and identified candidate bio-
arthritis. markers (TABLE 1). We first briefly discuss the anatomy
• Synovial biopsy is a safe and well-tolerated procedure that is becoming more widely and physiology of the healthy synovial joint, the main
available. changes that occur in the inflamed joint, and current
• There is a significant body of work from the past 30 years analysing the cellular and approaches to biopsy retrieval and analysis.
molecular changes in synovial tissue from patients with rheumatoid arthritis to
identify specific biomarkers. Synovial joint anatomy and physiology
• Technological advances in molecular and cellular analysis now provide new The synovial joint comprises opposing bones with artic-
opportunities for defining new biomarkers and targets. ular surfaces that are covered by cartilage. The main
protein in bone is type I collagen, whereas cartilage com-
prises mainly type II collagen and proteoglycan mole-
cules. The non-articulating surfaces of synovial joints
Author addresses are lined by a thin adventitious layer known as the syn-
1
Centre for Arthritis and Rheumatic Disease, University College Dublin, Dublin
ovium. Normal synovial tissue comprises one to three
Academic Medical Centre, St. Vincent’s University Hospital, Elm Park, Dublin 4, layers of specialized columnar FLSs that are interspersed
Ireland. with macrophages12. The entire structure is enclosed by
2
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, a fibrous capsule and, together with ligaments, muscles
Av. Prof. Egas Moniz, 1649–035, Lisbon, Portugal. and tendons, the fibrous capsule confers strength and
3
University of California San Diego School of Medicine, 9500 Gilman Drive, La Jolla, stability to the joint.
California 92093, USA. Several factors contribute to the maintenance of
4
Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton normal homeostasis in the synovial joint, including the
Hospital, Chapeltown Road, Leeds LS7 4SA, UK. expression of the protective lubricin13, the secretion of
5
Rheumatology Research Group, University of Birmingham, Edgbaston, Birmingham matrix metalloproteinases (MMPs) by FLSs, the immune
B15 2TT, UK. sentinel roles of resident macrophages and FLSs, the reg-
6
Arthritis Unit, Rheumatology Department, Hospital Clínic, IDIBAPS, Villarroel 170, ulated entry and exit of leukocytes involved in immune
08036 Barcelona, Spain. surveillance, and local regulation by cytokines and
7
Rheumatology Unit, Department of Medicine (Solna), Karolinska Institute and growth factors.
Karolinska University Hospital, 171 76 Stockholm, Sweden. Cytokines and growth factors are important regula-
8
Cardiff University School of Medicine, Institute of Infection and Immunity, 1 st Floor, tors of FLSs and chondrocytes14–16. Cytokines are catego-
Tenovus Building, Heath Park, Cardiff CF14 4XN, UK. rized as either pro-inflammatory or anti-inflammatory
9
Department of Molecular Rheumatology, Trinity College Dublin, University of Dublin, depending on their immediate effects on specific tissues,
College Green, Dublin 2, Ireland. although considerable potential exists for pleiotropism
10
Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology depending on the cells targeted and the microenviron-
and Immunology Centre, Academic Medical Centre, University of Amsterdam, ment. Cytokines and growth factors are ubiquitous in the
Room F4‑105, PO Box 22700, 1100 DE, Amsterdam, Netherlands. synovium and synovial space, and originate either from
11
Clinical Unit Cambridge, GlaxoSmithKline, Cambridge, UK. the plasma or from FLSs, chondrocytes and cells in the
12
Centre for Experimental Medicine and Rheumatology, John Vane Science Centre, surrounding tissues16.
William Harvey Research Institute, Barts and the London School of Medicine and The joint is a dynamic environment that is subject to
Dentistry, Queen Mary University of London, Charterhouse Square, London minor trauma continually — owing to movement and,
EC1M 6BQ, UK. in some joints, compression due to weight bearing —
13
Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, and is therefore subject to continued wound healing and
Framlington Place, Newcastle upon Tyne NE2 4HH, UK. repair processes. Continual remodelling of the articular
14
Department of Medicine, Svendborg Hospital, Odense University Hospital, cartilage and adjacent bone is therefore necessary, and
Valdemarsgade 53, 5700 Svendborg, Denmark. this process requires a balance of anabolic and catabolic
15
Université catholique de Louvain and Department of Rheumatology, Cliniques enzyme activity in both cartilage and bone. Carefully
Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Bruxelles, Belgium. regulated proteolytic enzymes are responsible for main-
16
Rheumatology Unit, Nantes University Hospital, Hôtel-Dieu, 1 place Alexis-Ricordeau, taining the balance between anabolic and catabolic pro-
44093 Nantes cedex 1, France. cesses within the joint and cartilage17. The collagenases
17
Rheumatology and Translational Immunology Research Laboratories (LaRIT), MMP1 (interstitial collagenase), MMP3 (strome-
Division of Rheumatology, IRCCS Policlinico San Matteo Foundation/University of lysin‑1), MMP8 (neutrophil collagenase), MMP13
Pavia, P.le Golgi 19, 27100 Pavia, Italy.
(collagenase 3) and MMP18 (also known as MMP19)
18
Institute of Infection, Immunity and Inflammation, College of Medicine, are the most important of these enzymes, as they are the
Veterinary and Life Sciences, University of Glasgow, 120 University Avenue,
only known enzymes that can directly cleave collagen at
Glasgow G12 8TA, UK.
a neutral pH18, but other MMPs contribute to collagen
19
Rheumatology, Flinders University, GPO Box 2100, Adelaide 5001, South Australia,
degradation once its triple helix structure has become
Australia.
unravelled19.
20
GlaxoSmithKline, Cambridge, UK.
Serine and cysteine proteinases are required to acti-
21
Institute for Molecular Life Sciences, RadboudUMC, Theodoor Craanenlaan 11,
vate pro-MMPs (that is, MMP precursors) after they are
Nijmegen 6525 GA, Netherlands.
secreted. Furthermore, inhibitors of these proteinases

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Box 1 | Synovial tissue research and the omics approach
The widespread availability of synovial tissue biopsy procedures and analytical methods
throughout the world52 will inevitably enable a targeted approach to the identification
of synovial biomarkers. The advent of new proteomic, transcriptomic and genomic
technologies, and the ability to combine clinical and radiological markers with these
technologies, will facilitate progress in this area. The omics approach has been usefully
applied to the identification of key players and protein interactions in several diseases.
Studying the genome, RNA expression or protein expression each have different biases,
and combined approaches could arguably lead to a more accurate assessment of
important protagonists104.
Proteomics offers the advantage that the functional units (that is, the proteins) of the
cell are being studied directly, and this approach is likely to provide information that
most accurately reflects what is actually happening in the synovium. Technologies
such as SomaLogics that have the power to measure thousands of proteins in small
volumes of tissue have the potential to enable a more complete characterization of
the protein networks that underlie diseases such as rheumatoid arthritis (RA)160.
Furthermore, new technologies for protein separation, processing and identification
are expected to increase proteome coverage. In RA, the proteomics approach has so
far focused on peripheral blood mononuclear cells, serum and synovial fluid66,67,139; the
possibility that the synovial tissue itself might hold the key to elucidating the
underlying disease-associated protein networks has yet to be fully exploited by
proteomic studies.
In relation to transcriptomic analysis, microarray technology has, to date, been the
most frequently used strategy in the field of biomarker research. This technology
facilitates the identification of candidate genes that are involved in pathophysiological
processes. However, gene expression levels do not always predict protein levels
owing to transcriptional and translational regulatory mechanisms and the activity of
protein-degradation processes17.
Microarrays contain probes for thousands of different genes, and they are suitable
for screening large cohorts; however, the high-throughput techniques used in
transcriptomics also enable the detection of significant gene-expression differences
within modestly sized cohorts161. Transcriptomic analysis is already being used to
examine the gene signatures of synovial tissue, and such investigation is augmented
by the newer sequencing technologies that enable deeper transcriptional coverage
than do microarrays and that include spliced variants. Several studies have
demonstrated that microarray technology is a useful and practical methodology for
studying gene expression in RA, and have characterized gene-expression patterns in
the synovia of patients with RA (see the section of the main text entitled
‘Gene-expression profiles’)162,163.
(such as tissue inhibitors of metalloproteinases (TIMPs)
and inhibitors of serine proteinases (SERPINs)) are also
present in the normal joint 20. The levels and activity of
these enzymes can be monitored indirectly by measuring
their degradation products in the synovial fluid21.
Features of the inflamed joint
The inflamed synovium has been studied at the macro-
Intimal lining layer scopic, microscopic and molecular levels. The synovium
The lining of the synovium is the primary target of disturbed immunomodulatory
comprising a few cells without pathways in RA. Rheumatoid synovial tissue appears
a basement membrane and
which covers the nonarticular
to be macroscopically hyperplastic and hypervascular
surface of the joint capsule. (FIG. 1a,b), while microscopic analysis reveals hyper­plasia
of the intimal lining layer (FIG. 1c) and the accumulation of
Synovial sublining inflammatory cells (FIG. 1d), including T and B lympho-
A loose connective tissue that
cytes, plasma cells, macrophages, neutrophils, mast cells,
lies beneath the intimal lining
of the synovium. natural killer cells and dendritic cells, in the synovial
sublining22. Like the target organs in other autoimmune
Pannus diseases (for example, Sjögren syndrome and auto­
A ‘tumour-like’ mass of immune thyroiditis), infiltrating T cells and B cells have
hyperplastic synovial tissue
that expands into the joint,
been demonstrated to form aggregates that have vary-
invading into bone and ing degrees of organization and the potential to produce
cartilage. disease-­specific ACPAs23,24.
Angiogenesis accompanies this immune cell accumu-
lation, but it occurs in an abnormal manner that results
in different patterns of blood vessels in different inflam-
matory arthropathies25. The new blood vessels seem to
be in an immature state26. They permit increased leuko-
cyte migration, and the synovial tissue transforms into
an invading pannus that can cause cartilage and bone
destruction27,28. Despite the increased vascular supply,
marked hypoxia has been demonstrated in inflamed syn-
ovial membranes in vivo29. The low tissue partial oxygen
pressures in inflamed synovial joint tissue are associated
with significant increases in macroscopic synovitis scores
and markers of microscopic inflammation, such as CD68+
macrophages and CD3+ T cells in the sublining, and var-
ious pro-inflammatory mediators (including TNF, IL‑1β,
IFNγ and the chemokine macrophage inflammatory pro-
tein 3α (MIP3α; also known as CCL20)). When primary
synovial fluid cells are exposed in vitro to partial oxygen
pressures similar to those in inflamed joints, cell migra-
tion is significantly increased, suggesting that hypoxia
drives pathological changes in the synovium26.
Many of the pathological changes in inflamed synovial
tissue are reflected in the synovial fluid, which has also
been studied intensively. Inflammation alters the perme-
ability of synovial tissue30; in the RA‑affected synovium,
the permeability to large molecules is increased, but that
to small molecules (for example, urea and glucose) is
decreased, an effect that is attributable to a combination
of increased vessel permeability, cellular infiltration and
synovial hyperplasia. As a result, the total protein content
of synovial fluid is higher during inflammation than in
the steady state. The molecular weight distribution of the
lubrication macromolecule hyaluronic acid is also altered
during inflammation, with a shift towards lower molecular
weight forms in RA31. In addition, rheumatoid joints show
increased loss of hyaluronic acid compared with healthy
joints, and the mean hyaluronic acid concentration is
lower in synovial fluid samples from patients with osteoar-
thritis (OA) or RA than in those from healthy controls32,33.
Synovial fluid samples from patients with inflamma-
tory arthritides have markedly raised cytokine concentra-
tions34. The role of cytokines in initiating and perpetuating
the synovial inflammatory response continues to be stud-
ied intensively, and has already led to the development
of several useful therapeutic agents and to the identi-
fication of further potential targets16. Changes in the
cellular infiltrate of RA‑affected synovial tissue have
long been recognized to be associated with the clinical
course of disease, and have been used to identify specific
responses to conventional and biologic DMARDs35–37.
In summary, the synovial tissue of patients with
inflammatory arthritides displays numerous alterations
relative to healthy synovial tissue. Thus, the study of syn-
ovial tissue is crucial to improving our understanding of
these diseases.
Synovial biopsy
The utility of synovial biopsy is clear; the analysis of
biopsy-obtained synovial tissue samples has increased
our understanding of the pathogenesis of RA, identified
potential therapeutic targets, and enabled the evaluation

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Table 1 | Areas in which synovial tissue research has provided insights into rheumatoid arthritis
Research areas Examples of key findings Refs
Pathogenesis of RA Fibroblast-like synoviocytes in RA‑affected joints have a distinct DNA methylation pattern, and express 77
genes involved in the JAK–STAT pathway and HOX genes
CD68+ macrophages are central effector cells in RA 118
CD3+CD45RO+ TH17 cells are pivotal in RA 72
CD20 CD22 B cells produce antibodies and cooperate with T cells in RA
+ +
23
Synovial biomarkers of early JNK activation is increased in early RA but not in undifferentiated arthritis 101
arthritis
Synovial CD22 and CD38 expression distinguishes patients with RA from those with non‑RA disease 99
Synovial biomarkers that Numbers of CD68+ macrophages, CCR7+ T cells and CD20+CD22+ B cells correlate with 116,121,136
correlate with treatment treatment-induced changes in disease activity
response and disease
severity Levels of ICAM1, MMP1 and OPG correlate with treatment response 40,48,57
Levels of S100A8, S100A9 and S100A12 correlate with the severity of joint erosion 139
TIE2 expression is higher in erosive disease than in self-limiting disease 100
Synovial biomarkers of RA Reduced numbers of CD68+ macrophages are found in patients in remission 121
remission
CCR7, CC‑chemokine receptor 7; HOX, homeobox; ICAM1, intercellular adhesion molecule 1; JAK, Janus kinase; JNK, JUN N‑terminal kinase; MMP1, matrix
metalloproteinase 1; OPG, osteoprotegerin; RA, rheumatoid arthritis; STAT, signal transducer and activator of transcription; TIE2, tyrosine kinase with Ig and EGF
homology domains 2; TH17, T helper 17.

Arthroplasty
Surgical reconstruction or
replacement of a synovial joint.
Arthroscopic biopsy
Minimally invasive procedure
to examine a synovial joint
using an endoscope.
of current and new treatments35–57. Synovial tissue ana­
lysis might also provide insights into the mechanism
of action of a given agent 58. This section discusses how
synovial biopsy and analysis are carried out, and sum-
marizes the main areas in which synovial tissue analysis
has proven informative.
Retrieving synovial tissue samples
Synovial tissue can be obtained by needle biopsy,
arthroplasty, arthroscopic biopsy, or using ultrasound to
guide the biopsy needle or grasping forceps58 (FIG. 2).
Arthroscopic biopsy enables the direct visualization
of the synovium, and the operator can select an area of
synovium to biopsy. By contrast, ultrasonography ena-
bles the indirect visualization of synovial thickness, and
synovial vascularity can simultaneously be assessed
by Doppler ultrasonography when selecting a suitable
biopsy site. Although blind biopsy has been validated,
arthroscopic biopsy and ultrasound-guided biopsy pro-
cedures are favoured by the majority of investigators
for proof‑of‑concept experiments, as sampling is more
specific for synovial tissue than connective tissue using
these methods52.
Arthroscopic and ultrasound-guided biopsy pro-
cedures are safe and well-tolerated. Data from 15,682
arthroscopies performed by rheumatologists revealed
a complication rate of 0.9% for haemarthrosis, 0.2% for
deep vein thrombosis, and 0.1% for both wound infec-
tion and joint infection59. These data were reproducible
at other centres, and the overall complication rate was less
than 0.3%. Similarly, a systematic review reported an over-
all major complication rate of 0.4% for ultrasound-­guided
biopsy procedures60.
Synovial tissue analysis
Questions remain about the best location from which
to obtain a biopsy sample within a given joint. In par-
ticular, concerns have been raised that mediators of
inflammation might be differentially expressed in differ-
ent parts of the same joint, particularly in the cartilage–
pannus junction (CPJ) versus non-CPJ sites, which are
known to behave differently 61. However, the numbers of
T cells62,63 and plasma cells63, and the expression levels
of several MMPs63 and granzymes63, are reported to be
similar in biopsy samples from CPJ and non-CPJ sources.
One study did find a difference for macrophages64,
but other studies did not replicate this finding 62,63.
Studies examining the optimal number of synovial tis-
sue specimens required for reproducible research stud-
ies suggest that at least six biopsy specimens should be
obtained58,65.
Although immunohistochemical analysis of syn-
ovial tissue (FIG. 3) has a minor clinical role in the dif-
ferential diagnosis of arthritis (for example, infectious,
granulomatous, infiltrative diseases or crystal arthrop-
athies), the identification of biomarkers that could be
used for diagnosis or for predicting disease progression
and response to treatment remains an unmet challenge.
Therefore, studies of the synovium have expanded
beyond immunohistochemistry to involve methods of
tissue digestion, homogenization and whole-tissue cul-
ture (FIG. 4). Methods of examining synovial tissue at a
molecular level include detailed omics technologies
(BOX 1). For such analysis, the synovial tissue obtained
from the joint is placed on saline-dampened gauze,
snap-frozen in the cryoprotective optimal cutting tem-
perature (OCT) compound or placed directly into an
RNA-stablizing solution (such as RNAlater).
Synovial fluid samples are centrifuged, and a cell pel-
let can be isolated or separated using a Ficoll gradient to
provide synovial fluid mononuclear cells. Several prog-
nostic biomarkers of RA have been identified in synovial
fluid and validated in serum samples66. Studies using this
strategy first identified proteins that were of potential
interest in the synovial fluid, and then searched for anti-
bodies to these proteins in the plasma67. The approach of

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a b effect72. Indeed, studies of synovial fluid and synovial tis-

c d
Figure 1 | The macroscopic and microscopic appearance of rheumatoid synovial
Nature Reviews | Rheumatology
tissue. Macroscopic images of synovial tissue from a patient with rheumatoid arthritis
demonstrating inflamed and hyperplastic synovial villi (part a) and hypervascularity
(part b). Representative microscopic appearance, stained with haematoxylin and eosin,
demonstrating the cellular infiltrate and lining layer hyperplasia thickness (indicated by
the black line) (part c) and representative Factor VIII immunostaining of the rheumatoid
synovium demonstrating increased synovial blood vessels (part d) (original
magnifications ×10).
obtaining and analysing different types of samples from
the same patient might be useful in future experiments
of synovial tissue, and the results of such research might
be more easily translated into clinical practice, as serum
samples can be obtained in a relatively non-invasive
manner. It is important to note that although synovial
fluid might reflect the synovial compartment better than
does blood, it still provides only indirect information,
and therefore studies of synovial tissue are essential68.
Although most research studies of synovial tissue biop-
sies have involved patients with RA, which is the focus of
this Review, synovial tissue sampling might also be useful
in the context of other inflammatory arthropathies such
Ex-TH17 cells as psoriatic arthritis68–71.
T helper 17 (TH17) cells can
switch to become ex-TH17 cells Main areas of progress
that no longer produce IL‑17 As mentioned above, synovial tissue research has fuelled
but have the ability to produce
IFNγ.
progress in several key areas; these areas are summarized
in TABLE 1 and discussed in more detail here.
Positional memory
Cells might demonstrate Insights into the pathogenesis of synovitis
different DNA ‘fingerprints’
The importance of directly analysing synovial tissue —
depending on the site of the
body at which they reside. the target tissue in RA — is evident from studies investi-
gating the pathogenesis of RA. For example, T helper 17
Undifferentiated arthritis (TH17) cells are expanded in the blood of some patients
Inflammatory oligoarthritis or with RA, and this finding provided the rationale for clin-
polyarthritis that does not
conform to any of the
ical trials of anti‑IL‑17 monoclonal antibodies; however,
recognized inflammatory as limited TH17 expansion occurs within the synovium
arthritis types. of patients with RA, this therapeutic approach had little
sue from patients with RA have shown an enrichment of
so‑called ex-TH17 cells at the site of inflammation73. This
finding might explain the failure of anti‑IL‑17 therapy
in some patients as the differentiated T cells no longer
produce IL‑17. Further emphasizing the importance of
direct synovial tissue analysis is the fact that no circulat-
ing biomarkers have yet been identified that can provide
a readout of the activity of the primary invasive cells in
RA, the FLSs74.
Synovial tissue analysis has also revealed some sur-
prising findings regarding pathogenic mechanisms
involved in RA. One study of paired biopsy samples
taken from the inflamed knee joint and an inflamed
small joint of patients with RA demonstrated similar
mean cell numbers for all markers investigated in the
synovial sublinings of both tissues75. Of further note,
patients with clinically evident disease that manifests at
small joints have been shown to have similar — albeit
less pronounced — abnormalities in clinically unin-
volved knee joints64,75,76. However, hyperplasia of the
intimal lining layer seemed to depend on local pro-
cesses; different joints showed no similarity in terms
of the numbers of intimal macrophages or FLSs 64.
Consistent with these findings, in RA, the FLSs from
different joints of the same patient show distinct DNA
methylation and transcriptome signatures, as well as
differences in FLS invasiveness, depending on their
positional memory77,78.
Early arthritis and disease stratification
Since 2002, cohorts of patients with early arthritis have
been gathered, and have provided clinical, histologi-
cal, DNA-level, mRNA-level and proteomic data; such
cohorts represent instrumental resources for investigat-
ing early disease79. Synovial tissue analysis is beginning
to have an impact on our understanding of early arthritis.
Although some progress has been made in terms of diag-
nosing RA earlier, signs of joint destruction can already
be present at the time of diagnosis and so developing our
understanding of early disease is important 80. We know
today that early, aggressive treatment is more successful
than is delayed treatment 81,82, and a ‘window of opportu-
nity’ is suggested to exist, during which RA can be most
successfully treated. Therefore, the use of biomarkers to
secure a diagnosis as early as possible will enable treat-
ment in the most timely manner and will secure the best
outcomes83. Patients with undifferentiated arthritis might
benefit most from early diagnosis. Although ACPA
detection is reasonably specific (96%), the diagnos-
tic sensitivity of ACPAs in early arthritis is 57%81, and
up to 30% of patients with RA never develop ACPAs,
highlighting the need for alternative biomarkers84,85. An
association has been defined between the presence of
circulating ACPAs and the subsequent development
of RA in individuals with arthralgia86, and of bone ero-
sions in patients with early untreated arthritis87. However,
a positive ACPA status in those with arthralgia is associ-
ated with the subsequent development of arthritis in only
20–30% of individuals after 30 months of follow-­up88,89,
further emphasizing the need for additional biomarkers.

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a b The analysis of synovial tissue samples from patients

c d
Figure 2 | Synovial tissue retrieval methods. a | NeedleNature
arthroscopy of |the
Reviews knee joint.
Rheumatology
b | Macroscopic image of synovial tissue biopsy using grasping forceps visualized by
arthroscopy. c | Ultrasound-guided biopsy. d | Representative image of an
ultrasonography scan.
A delay in diagnosing RA could arise from either a
lack of a definitive biomarker or a failure to meet current
diagnostic criteria, and these criteria have a considerable
reliance on biomarkers; thus, further research into spe-
cific susceptibility biomarkers is warranted. Two studies
published since 2015 have identified circulating bio-
markers of RA in patients who lack detectable circulat-
ing ACPAs; this subset of patients is an important group
to study, and data from these patients might contribute
greatly to our understanding of disease pathogenesis90,91.
Synovial tissue analysis could be key to the identification
of the required biomarkers.
Cohorts of individuals who are at risk of develop-
ing arthritis have been the subject of much research.
One potential corollary of such studies is the promise
of a cure for RA, or a preventive approach that could
detect and therapeutically target the initial breach of
self-­tolerance92. The synovial tissue of patients who are
at risk of arthritis has been examined in two relatively
small studies. Little evidence of synovitis was found in
Disease stratification the first study88, and subtle T cell infiltration was noted
The concept that a disease in the second89. Further study of synovial tissue samples
can be classified into distinct from at‑risk individuals is required, as is the analysis
subsets that exhibit of other tissues, such as the lung and lymph nodes,
differential outcomes and
responses, and that can each
which might be important in the very early stages
be labelled by a biomarker or of arthritis as they are the first sites at which antigen
a combination of biomarkers. is presented93,94.
with early RA has provided important insights. In initial
studies, the synovia of patients with early disease have
shown few molecular differences when compared with
synovia of patients with late disease23,95. However, a study
published in 2012 identified a highly expanded, specific
T cell clone in the synovia of patients with early RA,
which underlines the importance of T cells in early-stage
disease96. Another study has indicated that epigenetic
changes occurring in FLSs over time might define the
different stages of RA after clinical onset 97. Furthermore,
in a preliminary report published in 2016, synovial
tissue obtained by ultrasound-guided biopsy from
unselected treatment-naive patients with early arthri-
tis showed increased expression of the macrophage-­
derived chemokines CXC-chemokine ligand 4 (CXCL4;
also called platelet factor 4) and CXCL7 (platelet basic
protein) only during the first 3 months of symptomatic
arthritis and not later in the disease98.
In addition to identifying potential pathogenic
mechanisms, synovial tissue biopsy might be useful for
informing differential diagnosis in early inflammatory
arthritis, as suggested by a study in which synovial CD22
and CD38 expression could distinguish patients with
RA from those with non‑RA disease99. The use of syn-
ovial biomarkers for early disease stratification was also
reported in a study of 50 patients with early arthritis who
had undergone synovial biopsy at inclusion and were
followed for 2 years100. The focus was on the angiogenic
processes involved in the initiation and perpetuation of
synovial inflammation, in particular vascular endothe-
lial growth factor (VEGF) and angiopoietins 1 and 2,
and their tyrosine kinase receptors VEGFR and tyros-
ine kinase with Ig and EGF homology domains 2 (TIE2;
also known as angiopoietin 1 receptor). The expression
of TIE2 was significantly increased in the synovia of
patients with erosive disease compared with the synovia
of patients who had self-limiting disease, and the expres-
sion of activated, phosphorylated TIE‑2 was significantly
increased in patients with persistent non-­erosive disease
or persistent erosive disease compared with patients
who had self-limiting disease. In addition, the activa-
tion of JUN N‑terminal kinase (JNK) is elevated in the
synovia of patients with early RA relative to the synovia
of patients with undifferentiated arthritis, before the
classification criteria for RA are met 101. Together, these
studies indicate that synovial tissue analysis can provide
information relevant to disease diagnosis.
Only a limited number of studies have analysed syno-
vial tissue from patients with early RA, and so the use of
synovial tissue markers in early diagnosis is clearly still
evolving. Although more research is needed, these stud-
ies suggest that a synovial biopsy at disease presentation
could be a useful tool for both patients and physicians, as
it could enable the stratification of early RA into short-du-
ration, self-limiting disease (which may be erosive or
non-erosive) versus severe, persistent and destructive
inflammatory disease, thereby informing the most appro-
priate treatment strategy102,103. This personalized medicine
approach tailors treatment on the basis of biomarkers
and so‑called ‘disease signatures’ (REF. 98), which enable

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a b antirheumatic treatments112,113. In addition, the number

of lymphocyte aggregates is reported to be predictive of
the clinical response to infliximab treatment 112,114.
Positivity for lymphocyte aggregates increased the
power of a prediction model that included baseline
disease activity evaluated by 28‑joint disease activ-
ity score (DAS28), ACPA positivity and synovial TNF
expression112. Taken together, these studies suggest that
although lymphoid aggregates may not enable the strat-
ification of disease into subtypes, they might represent a
biomarker of treatment response.

Lymphocytes. Simple cell counts (or cellular infiltrates)

Figure 3 | Synovial tissue immunostaining. Representative
Nature Reviews
images | Rheumatology
of biopsy-obtained
rheumatoid synovial tissue demonstrating CD19+ B cell lymphoid aggregate (part a) and
CD3+ T cells (part b). Original magnifications ×10.
disease stratification104,105. The sensitivity and specificity
of disease stratification could theoretically be improved
by using a combination of biomarkers. For example, a
positive clinical response of RA to anti-TNF treat-
ment with etanercept has been predicted using a bio-
marker signature comprising 13 autoantibodies and 11
cytokines. This study included three ethnically distinct
populations, and for North Americans it demonstrated a
positive predictive value of 71%, although independent
validation is required11.
Disease stratification is important as therapies are
commonly selected on a trial-and-error basis but less
than 50% of patients with RA experience a 50% improve-
ment in their arthritis in response to any single biologic
therapy 106–108. In the time that an ineffective treatment is
administered, the disease might progress, and patients
could potentially experience unnecessary adverse events.
Therefore, biomarkers that predict response to a given
treatment will be of great clinical utility. Synovial bio-
markers are likely to be of the greatest clinical utility,
and a great deal of work has concentrated on studying
features of the inflamed, RA‑affected synovium before
and after treatment. Examples of such studies, and others
that have analysed the ability of synovial tissue biomark-
ers to predict disease prognosis and response to therapy,
are summarized in the next section.
Different types of synovial biomarker
Lymphocyte aggregates. A detailed discussion of lym-
phocyte aggregates is beyond the scope of this Review,
and the topic has recently been discussed in detail else-
where109; however, we wish to briefly highlight the poten-
tial biomarker role of these structures here. A number of
studies have addressed whether lymphocyte aggregates
of synovitis are associated with clinical phenotype or the
development of persistent, erosive disease. In two large
studies, lymphocyte aggregates were found in approxi-
mately 30% of patients with established RA but did not
associate with a clinical phenotype110,111. Similarly, the
presence of lymphocyte aggregates in patients with early
arthritis did not predict an aggressive disease course,
and aggregates were rapidly diminished by several
were recognized as RA‑associated synovial tissue bio-
markers more than 20 years ago. In a study published
in 1989, T cell numbers were shown to decrease after
at least 6 months of gold treatment, and the ratio of TH
cells to suppressor T cells or cytotoxic T cells was found
to be reduced in patients who were treated successfully 35.
Furthermore, the number of biopsy samples in which
B cells could be identified decreased from 36% before
successful treatment to 7% after treatment 35.
Further evidence that the abundance of synovial lym-
phocytes (as assessed by staining for cell markers) rep-
resents a biomarker of treatment response comes from
studies of the following RA therapies: 16 weeks of metho­
trexate, which caused a decrease in the synovial expres-
sion of markers of T cells (CD3 and CD8) and plasma
cells (CD38)115; 4 weeks of infliximab, which reduced
synovial CD3+ T cell numbers in patients showing a clin-
ical response38; 2 weeks of infliximab, which reduced the
numbers of synovial CD3+ T cells and CD22+ B cells44;
2 weeks of prednisolone, which reduced the numbers of
synovial CD4+ T cells, CD5+ B cells and CD38+ plasma
cells, as well as the number of CD55+ FLSs41; and various
durations of rituximab treatment, which partially but
not invariably depleted synovial B cells, with reductions
in T cells and CD68+ macrophage numbers53,116,117. The
changes in CD68+ macrophage numbers after rituximab
treatment have been replicated independently in another
centre118. By contrast, one other group showed reduc-
tions in B cell numbers with minimal or no change in
macrophages and T cells119; this variation in findings is
possibly explained by differences in patient populations,
methods for immunohistochemistry or analysis such as
digital image analysis.
Macrophages. Although macrophages were not included
in the 1989 study described at the start of the previous
section35, the most convincing evidence for a cellular
biomarker of treatment response points to the macro­
phage marker CD68. This evidence comes from many
studies, including those of patients receiving the fol-
lowing RA therapies: 2 weeks of prednisolone, which
reduced CD68+ macrophage abundance in the synovial
sublining 41; 12 weeks of gold therapy, which was associ-
ated with an abundance of changes in all synovial layers
independently of the site of synovial biopsy 120; various
durations of treatment with methotrexate or gold121, for
which the reduction in CD68+ macrophage numbers
in the synovial sublining was particularly pronounced

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a b and sublining macrophages after active treatment,

c d
Figure 4 | Ex vivo synovial tissue culture viability. To establish ex vivo rheumatoid
synovial biopsies, whole synovial tissue can be transferred Nature Reviews
directly | Rheumatology
from the biopsy
forceps to culture medium. Synovial tissue can be maintained in culture for up to 72 h
and remains viable during this time. a | Representative photomicrographs of synovial
tissue, after 72 h in culture, stained with fluorescent calcein green indicating viable cell
nuclei (white arrows). b | Viable blood vessels in cultured synovial tissue (white arrow).
c,d | Representative photomicrographs of synovial tissue, after 72 h in culture, embedded
in optimal cutting temperature (OCT) compound and stained with haematoxylin and
eosin to demonstrate the intact structural morphology of the synovial tissue: intact lining
layer, sublining layer and blood vessels (black arrows). Original magnifications ×10.
in those who showed a clinical response according to
ACR criteria; 16 weeks of treatment with leflunomide
or methotrexate, which were specifically associated
with abundance changes in the synovial sublining and
the intimal lining layer, respectively 40; various durations
of infliximab treatment, which reduced CD68+ macro­
phage numbers in the synovial sublining 44,122; anakinra,
over 24 weeks, which reduced the size of the intimal
CD68+ macrophage population42; and various durations
of rituximab treatment, which reduced the abundance
of intimal lining CD68+ macrophages in responders53.
One study has systematically investigated the utility
of synovial sublining-localized CD68+ macrophages
as a candidate biomarker across different interven-
tions and kinetics, and found that changes in the num-
bers of these cells correlate with clinical improvement
independently of the therapeutic strategy; the number
of CD68+ macrophages decreased as disease activity
reduced (as measured by DAS28), thus demonstrating
that such cell counts could be used as a biomarker of
therapeutic response123. This finding was confirmed in
a multicentre study that reported excellent intercentre
agreement 118. Furthermore, the sensitivity to change
of synovial CD68 expression is good for both DAS28
including rituximab124; in addition, it has been shown
that DAS28 is more susceptible to placebo effects than
synovial CD68 expression125. Therefore, while we do not
propose to focus on synovial biomarkers without clin-
ical assessment, using this biomarker has been shown
to be less susceptible to the placebo effect and expec-
tation bias123,125. This work has led to the development
of a simple decision tree to inform ‘go/no‑go’ deci-
sion-making in drug development, which incorporates
clinical assessment, mechanism of action and synovial
CD68 expression and has been used in the evaluation of
numerous compounds since its proposal51. In a ballot at
the Outcome Measures in Rheumatology (OMERACT)
9 conference general assembly in 2008, 59% of the del-
egates agreed that CD68 expression in synovial tissue
is less susceptible to a placebo effect and expectation
bias than clinical evaluation, compared with 13% who
disagreed118.Therefore, substantial evidence exists to sug-
gest that synovial CD68 expression in synovial sublining
macrophages demonstrates validity, reliability and feasi-
bility as a biomarker of disease activity and could there-
fore be used to assess the therapeutic efficacy of novel
treatments118,123,125,126. All of these studies have used the
same standardized techniques of immunohisto­chemistry,
which have been extensively validated across multiple
EULAR European Synovitis Study Group centres118.
By contrast, three studies from the same centre
reported minimal or no change in macrophage cells,
possibly owing to the use of different methology; in
studies of rituximab119, abatacept 127 and, more recently,
the signal transducer and activator of transcription
(STAT) inhibitor tofacitinib128 some reduction in sub-
lining macrophages was apparent but this reduction was
not statistically significant. A proof‑of‑concept study of
a CC-chemokine receptor 1 (CCR1) antagonist, used at
high doses to achieve high levels of receptor occupancy,
did show a reduction in macrophages, CCR1+ cells and
a trend towards clinical response129. Additionally, in
the single study in which similarly high levels of CCR1
receptor occupancy were achieved there was clear evi-
dence of clinical efficacy 130, supporting the predictive
value of this approach.
Cytokines. As mentioned in the ‘Features of the inflamed
joint’ section, the increased expression of several
cytokines in inflamed synovial tissue is well established.
Indeed, synovial TNF and IL‑6 concentrations correlate
with disease activity, independently of disease duration26.
With regards to the effects of treatment on cytokines,
the expression levels of IL‑1β and TNF were 40% (95% CI
18–56%) and 52% (95% CI 10–74%) lower, respectively,
following prednisolone therapy compared with placebo
treatment 41. Notably, this effect was mainly attributable
to changes in the synovial sublining, and seemed to cor-
relate with clinical improvement41. Significant changes in
cytokine expression have also been reported in the syno-
vial lining, perivascular tissue and connective tissue after
12 weeks of gold treatment35. In the intimal lining layer, the
levels of IL‑1α, IL‑1β and IL‑6 were significantly reduced
after treatment, and this reduction seemed to correlate

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with clinical response. TNF was also reduced in all three
areas, but the reduction in the synovial lining was not sta-
tistically significant35. In another study, TNF levels were
only slightly reduced in synovial samples from patients
who received 16 weeks of treatment with either metho-
trexate or leflunomide40. IL‑1β levels were only moder-
ately reduced in the leflunomide-treated patients, whereas
reductions in the methotrexate-treated patients were
significant, which potentially reflects the different mech-
anisms of action of these DMARDs40. Another study has
also reported that the expression of IL‑1β (but not that
of IL‑1α) is significantly reduced after 16 weeks of treat-
ment with methotrexate and found that this reduction
correlated with clinical response115.
As highlighted above, targeting cytokine signal-
ling pathways, for example the STAT pathway, is an
interesting and novel approach. Tofacitinib has shown
significant clinical benefit in patients with RA and is
associated with a significant reduction in expression of
phosphorylated STAT in synovial tissue, which suggests
that the level of phosphorylated STAT could be a useful
biomarker of response to this therapy128. Although not
itself a cytokine, acute serum amyloid A (A‑SAA) regu-
lates the expression of cytokines and is expressed in RA
synovial tissue, where it has a role in inducing angio­
genesis, cell–matrix interactions, and the expression of
chemokines and MMPs 131. Furthermore, blockade
of the A‑SAA receptors scavenger receptor class B mem-
ber 1 (SRB1)131 and Toll-like receptor 2 (TLR2) inhibits
FLS migration and invasion in synovial explants from
patients with RA132. Importantly, baseline serum A‑SAA
levels independently correlate with the 28‑joint swol-
len joint count and 1‑year radiographic progression in
patients with RA20. Therefore, serum A‑SAA is a prom-
ising biomarker of disease activity, warranting further
investigation of its expression in the synovia of patients
with RA133.
Chemokines. Leukocytes are attracted to target tissues
by soluble chemotactic cytokines termed chemokines,
which are released from activated cells in the tissue to
stimulate leukocyte migration through the endothe-
lial barrier 134. The chemokine monocyte chemotactic
protein 1 (MCP1; also known as CCL2), among others,
is expressed abundantly in both serum and synovial
tissue samples from patients with RA49. The develop-
ment of clinical signs of synovial inflammation in RA
is specifically associated with the increased synthesis of
CXCL8 (also known as IL‑8)135, and the expression
of both CXCL8 and MCP1 in synovial tissue (both the
lining and sublining) reflects response to therapy in
patients with active RA who have received infliximab;
the synovial expression of growth-regulated protein‑α
(GROα), RANTES (also known as CCL5) and MIP1β
(also known as CCL4) was also reduced but not to a
significant extent 44. Thus, chemokines could represent
a target and a biomarker of treatment response. Indeed, a
=proof‑of‑concept study of an oral CCR1 antagonist
in patients with RA showed a trend towards clinical
improvement and a concomitant, significant reduc-
tion in synovial macrophage numbers and chemokine
expression, suggesting that targeting CCR1 results in
changes that could also represent biomarkers of response
to this antagonist 129.
S100 proteins. Similarly to some cytokines and chemok-
ines, the S100 protein family — which comprises closely
related, low-molecular-weight (9–14 kDa) acidic calci-
um-binding proteins — have pro-inflammatory effects,
and they are overexpressed in inflammatory compart-
ments. S100 proteins are involved in calcium-dependent
cell activities such as cytoskeleton regulation, and cell
migration and adhesion, and they also have extracellu-
lar roles136. S100A8 (also known as MRP8) and S100A9
(also known as MRP14) regulate myeloid cell function
and control inflammation137, and S100A12 (also known
as MRP6) has important activities in relation to innate
and acquired immune responses138. One study using
quantitative proteomics demonstrated an association
between the severity of joint erosion in RA and the levels
of S100A8, S100A9 and S100A12 in both synovial fluid
and serum samples139; this potential role of S100 proteins
as synovial biomarkers requires further study.
Adhesion molecules. The expression of intercellular
adhesion molecule 1 (ICAM1) is significantly reduced
in patients with RA who are treated with either lefluno-
mide or methotrexate40. Notably, the significant decrease
in ICAM1 expression was seen only in those who
responded to treatment. The expression of vascular cell
adhesion molecular 1 (VCAM1) was reduced in both
treatment groups, but this reduction was significant only
in the leflunomide-treated patients40.
Another study demonstrated that the expression of
VCAM1 and E‑selectin was significantly reduced after
16 weeks of treatment with methotrexate, but in this
study the changes in ICAM1 expression did not reach
statistical significance115. Similarly, treatment with inflix-
imab has been shown to reduce VCAM‑1 and E‑selectin
expression in repeat biopsies taken 4 weeks after treat-
ment38. Interestingly, the effect of anakinra might be dose-
dependent, as patients taking a dose of 150 mg per day — but
not those receiving a lower dose of 30 mg per day — were
shown to have reduced synovial expression of E‑selectin,
ICAM1 and VCAM1 (REF. 42). Together, these stud-
ies highlight the biomarker role of synovial adhesion
molecule expression.
Mediators and products of bone, cartilage and synovial
tissue degradation. Serum levels of collagen biomarkers
and MMPs are known to predict radiographic progres-
sion in RA, and therefore could represent prognostic
biomarkers27,140, but could the synovial levels of these
molecules also be a biomarker?
Although MMPs are present in normal synovial
fluid, their concentrations are increased in synovial fluid
from patients with RA, psoriatic arthritis and OA17,141,142.
MMP1, in particular, might be a synovial biomarker of
treatment response, as suggested by a study reporting
that monotherapy with methotrexate or leflunomide
significantly reduced the expression of MMP1 and the
MMP1:TIMP1 ratio in synovial tissue samples from

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patients with RA after 4 months of treatment 40. Notably,
the changes were more pronounced in patients who
fulfilled the ACR20 response criteria40.
In addition to studies of matrix-degrading MMPs, a
number of studies have analysed the effects of immuno­
modulatory treatment on synovial mediators of bone
destruction. Treatment with either infliximab or etaner-
cept increases the expression of osteoprotegerin (OPG;
also known as TNFRSF11B) in synovial tissue, but had
no effect on the expression of receptor activator of nuclear
factor‑κB ligand (RANKL; also known as TNFSF11),
resulting in an increased OPG:RANKL ratio48. By con-
trast, rituximab induces a 99% decrease in the numbers of
receptor activator of nuclear factor‑κB (RANK)-positive
osteoclast precursors and 37% decrease in RANKL
expression, but only a nonsignificant reduction in syn-
ovial OPG expression143. However, not all RA therapies
induce changes in the levels of these bone-destructive
mediators; indeed, abatacept does not significantly
affect the synovial levels of mRNA expression of OPG,
RANK or RANKL127, suggesting that the biomarker role
of these synovial molecules might only be relevant in
specific settings.
Antigens and antibodies. The expression of antigenic
proteins has been described in synovial tissue samples
from patients with RA. For example, one study reported
that deiminated protein — such as the α- and β‑chains
of fibrin — present in RA‑affected synovia seem to be
major antigenic targets of ACPAs144. In addition, anti‑Sa
antibodies that recognize deiminated vimentin have
been isolated from RA‑affected synovia and seem to be
specific to RA145. Intracellular citrullinated proteins that
colocalize with ACPA reactivity have also been identified
in synovial tissue from patients with RA146, but the pres-
ence of citrullinated antigens in synovia is not specific
to RA24. Finally, anti-fillagrin antibodies are produced by
local plasma cells that are resident in the RA pannus147,
and thus could also be synovial biomarkers of RA.
Gene-expression profiles. Most of the previous discus-
sion has focused on protein-level data, predominantly
from immunohistochemical analyses; however, as men-
tioned above and discussed in BOX 1, studies have also
identified changes in synovial gene-expression profiles,
and these profiles could represent biomarkers. An exam-
ple of how transcriptomic data can be clinically useful
comes from a study that used these data to create a rule-
based classification that could differentiate between RA
and OA148. In addition, gene-expression variance among
patients with RA has been described for genes involved
in processes such as cell proliferation, cell survival,
angio­genesis and the regulation of inflammation149.
Several studies have investigated links between
gene-expression profiles and treatment response; for
example, genes involved in inflammation shown to be
upregulated in pretreatment biopsy-obtained synovial
tissue from patients with RA who subsequently responded
to anti-TNF therapy 150. In a larger follow‑up study, RNA
analysis of pretreatment synovial tissue from patients with
RA who were positive for lymphocyte aggregates revealed
that 38 transcripts were associated with clinical response
to infliximab treatment 114. A study of paired synovial
tissue samples taken from patients with RA before and
12 weeks after initiation of adalimumab treatment also
identified genes that were differentially expressed in sam-
ples from responders and non-responders151. These genes
could be split into two distinct families: genes involved in
the regulation of immune responses and genes involved
in the regulation of cell division. To confirm the micro­
array findings, the synovial expression of selected mol-
ecules was assessed using specific antibodies, and the
expression of IL‑7 receptor α‑chain (IL‑7Rα), CXCL11,
IL‑18, IL‑18 receptor accessory protein (IL‑18RAP) and
the proliferation marker MKI67 was found to be sig-
nificantly higher in poor responders than in moderate
and good responders. Thus, these findings link gene-
expression changes to protein-level changes and, consist-
ent with studies discussed above, they emphasize the role of
molecules involved in cytokine and chemokine signalling as
potential biomarkers of treatment response151.
Gene-expression analyses also support the role of
macrophages and T cells as biomarkers of treatment
response. For example, a study of paired synovial biop-
sies performed in patients with RA before and after
initiation of rituximab treatment revealed that clinical
responders demonstrated higher synovial expression
of macrophage-associated and T cell-associated genes,
whereas those with a poor clinical response showed
higher synovial expression of IFNα and genes associated
with matrix remodelling 152.
Challenges in biomarker identification
Most studies of serial synovial biopsies have been per-
formed on patients with known diagnoses and have aimed
to investigate response to treatment. There remains a crit-
ical need to identify diagnostic biomarkers that can be
used in clinical practice. Biomarkers could reduce the time
taken and patient numbers required to evaluate the poten-
tial efficacy of new drugs51,153. The number of patients with
active disease who are eligible to participate in studies is
limited. As with all trials, the number of patients who are
to be put at risk by exposure to drugs at an early stage of
drug development, as well as to be placed on placebo, are
restricted by ethical considerations154.
Although finding biomarkers in peripheral blood is
attractive because obtaining blood samples is more feasible
and less invasive than synovial biopsy, the inflamed syn-
ovium is the ultimate target of inflammation and should
thus be a rich source of potential biomarkers. Furthermore,
many confounding factors might interfere with periph-
eral blood profiles. Some authorities have suggested that
a more targeted approach to searching for serum markers
should first involve the identification of potential biomark-
ers in the inflamed synovial joint, and then the study of
these biomarkers in the serum155. Such an approach has
demonstrated clinical utility in patients with RA in a study
reporting that candidate peripheral biomarkers of synovial
pathotype predicted response to biologic therapy156.
New technologies are advancing synovial tissue ana­
lysis, but several issues remain to be addressed. Although
new technologies have enabled faster and more complete

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analyses of proteins, the high complexity of proteins and
protein isoforms in the synovial joint makes the inter-
pretation of ‘shotgun’ proteomic data challenging. The
interpretation of data from microarrays is also problem-
atic; for example, three widely used microarray platforms
have demonstrated poor reproducibility 157. In addition,
as array datasets contain high levels of background sig-
nals, they have decreased sensitivity to transcripts that
are present in low numbers158.
Cost and time are also important issues; for exam-
ple, the development of high-quality immunoanalytical
assays can be slow and expensive, which limits the ver-
ification of candidate biomarkers, despite the increase
in the availability of means to biopsy synovial tissue. In
addition, when data from control synovial tissue speci-
mens is available, OA is often used as a disease control,
although it is increasingly recognized that OA has an
underlying inflammatory response, albeit one that is lim-
ited and less associated with specific autoimmunity than
is the response that underlies RA159. These issues perhaps
explain why although a great many genomic biomarkers
exist that can predict response to treatment or who is most
at risk of adverse events in many areas of medicine, rheu-
matology seems to have experienced only limited benefit
from this emerging field. Future progress in identifying
genomic biomarkers, and other types of biomarker, will
probably be fuelled by synovial tissue analysis, which has
not yet been extensively performed in some areas; for
example, although many studies have attempted to iden-
tify gene-expression profiles that can predict response to
anti-TNF treatment, to our knowledge only those reported
above have used synovial tissue to search for these112,114,151.
Conclusions
Synovial tissue represents the target tissue of auto­
immune arthritides such as RA. New, safe methods of
obtaining samples of synovial tissue are becoming more
widely available. In this Review we have highlighted
those studies that analyse the cellular and molecular
characteristics of RA synovial tissue and how the results
have advanced the field in terms of patient stratifica-
tion, therapeutic target development and identification
of biomarkers of response to therapy. In addition, we
have reviewed the use of synovial tissue analysis as an
outcome measure for clinical trials in RA. Finally, the
future application of rapid advances in molecular tech-
nologies to synovial tissue analysis will probably lead to
major benefits for patients with RA.

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Acknowledgements
We wish to thank all our colleagues in the European Synovitis
Study Group and in the OMERACT group who have sup-
ported the development of synovial tissue research.
Author contributions
D.J.V., C.O., U.F., A.N., A.P. and J.E.F. researched data for the
article, made a substantial contribution to the discussion of
article content, wrote the manuscript, and reviewed and
edited the manuscript before submission. E.S., F.H., S.A.J., T.
McG. and R.T. researched data for the article, wrote the man-
uscript, and reviewed and edited the manuscript before sub-
mission. A.F. researched data for the article, and reviewed and
edited the manuscript before submission. B.R.L. wrote the
manuscript, and reviewed and edited the manuscript before
submission. D.L.B., M.H.B., C.D.B., J.D.C., A.I.C., E.H.C., P.E.,
D.G., J.D.I., B.L., A.M., I.B.M., C.P., M.S. and P.P.T. reviewed
and edited the manuscript before submission.
Competing interests statement
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

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